The present invention relates generally to flow cytometry. More specifically, it relates to the simultaneous measurement of incorporated BrdU and the total cellular DNA content by flow cytometric techniques.
The growth of any tissue at the cellular level is governed by variations in (1) the length of the cell cycle in the fraction of cells that continuously divide, (2) the growth fraction or the fraction of cells in the cell cycle at a given time and (3) the rate of cell loss. All these three mechanisms are important and operative in the growth of normal and malignant cells. A study of the cytokinetic properties of normal and tumor tissues, therefore, aid in the development of effective anticancer strategies for cell-cycle-phase-specific agents.
Cells in the body of an adult animal may be divided into three major categories: (1) differentiated cells i.e., cells which do not divide and proliferate and which eventually die; (2) cells that do not normally divide and therefore do not synthesize DNA but ca be induced to do so by an appropriate stimulus; and (3) continuously dividing cells which actively synthesize DNA and which move around the cell cycle from one mitosis to the next. The life cycle of a continuously dividing cell exhibits three distinct and consecutive phases. There is a period, the G.sub.1 phase, during which the cell is preparing for DNA synthesis but no actual, appreciable synthesis occurs. This phase is followed by the S phase, during which DNA is actively synthesized. During this phase, the cells take up nucleic acids present in the surrounding medium or environment, incorporating them into the DNA that is synthesized. The DNA content of the cell is doubled during the S phase. Following the S phase is the G.sub.2 M phase during which period the cell prepares for mitosis. No DNA synthesis occurs in the G.sub.2 M phase either.
The ability to measure, distinguish and differentiate between cell populations in the above three phases is, therefore, essential for many biological ad biomedical investigations. The cell cycle traverse characteristics of normal and malignant cells, measured as a function of the frequency of DNA synthesizing cells, yield valuable information concerning the treatment prognosis and therapy for various types of cancer. Studies of the cell cycle traverse of drug treated populations also yield valuable information about the mechanism of action of the drug.
S. C. Barranco et al., Cancer Res., 42, 2894 (1982), studied the cell kinetics properties of CHO cells to direct the timing of two-drug combination treatment of normal and tumor cells.
M. G. Pallavicini et al., Cancer Res., 42, 3125 (1982), measured the DNA distribution sequences, tritiated thymidine uptake by tumor cells and the radioactivity of cells labeled with tritiated thymidine prior to and after treatment with 1-beta-D-arabinofuranosylcytosine, a cytotoxic agent.
J. S. Hart et al, Cancer, 39, 1603 (1977), studied the prognostic significance of individual patient characteristics using eight pretreatment variables and reported that cytokinetic factors were important factors in the administration of chemotherapy and were of clinical importance in selecting approaches to therapy.
Coffino et al., Proc. Natl. Acad. Sci., 72, 878-882, (1975), studied the effect of dibutyryl cyclic AMP (dbc-AMP) on the cell cycle traverse of S-49 mouse myeloma cells by measuring DNA distributions and found that the induced cytokinetic changes yielded valuable information about the mechanism of action of the dbc-AMP.
The cell fraction in the S phase may be determined by measuring the uptake of radioactively labeled nucleic acids present in the environment. One of the most commonly used methods is the measurement of the uptake of labeled nucleic acids, such as thymidine, uridine or bromodeoxyuridine which are chemical analogs of thymidine and which are readily incorporated by the cell into its DNA. The presence of the label may be detected by autoradiography or liquid scintillation counting.
H. Quastler and F. G. Sherman, Exp. Cell Res., 17, 420 (1959) reported an analysis of the kinetics of a particular cell population, the different compartments existing in the system, transitions between the compartments which occur regularly, and estimates of the system parameters and individual variations. Their studies indicated that differentiation of the cells in their system occurred only during certain critical phases of the generative cycle.
Flow cytometry has been applied for the measurement of DNA in cultured cells to monitor ploidy of cells and changes in cell growth pattern (M. A. Van Dilla et al, Science, 163, 1213 (1968)).
J. W. Gray et al., in Cell Tissue Kinet., 10, 97 (1977) described a rapid method for the analysis of cell cycle traverse. According to this method, cells in S phase were pulse labeled with a radioactive DNA precursor and the progress of the labeled cells through the cell cycle was monitored by means of the label using flow cytometry and scintillation counting.
Recently, a method for the detection of BrdU incorporation using polyclonal antibodies has been reported by H. G. Gratzner et al., Exp. Cell Res., 95, 88 (1975); H. G. Gratzner et al., J. Histochem. Cytochem., 24, 34 (1976); H. G. Gratzner et al., Res. Comm. Chem. Pathol. Pharmacol., 20, 34 (1978).
Several measurement techniques, such a autoradiography, liquid scintillation counting, cytometric imaging, flow cytometry and the like, which have been developed over the years, suffer from serious limitations. Autoradiography is time consuming and involves laborious measurements. Furthermore, the method is also limited by the difficulty in distinguishing between unlabeled and weakly labeled cells and with background noise.
Scintillation counting which is based on the radiation emissions from the radioactive label, such as tritium, .sup.14 C, .sup.32 P and the like, requires measurements involving a small fraction of labeled cells in large cell populations and does not lend itself to single cell analysis or measurements. The use of antibodies offers an attractive alternative, but the polyclonal antibodies used exhibit variable specificity depending on the animal used for the immunization and also lack sufficient specificity. Furthermore, they cross-react with thymidine and are difficult to separate from the other immunoglobulins present in the system.
For the measurement of cell growth patterns, it is necessary to resolve the DNA distribution into its component parts. This analysis is not simple since the population distribution curves for the various phases of the cell cycle are, most often, quite complex due to considerable overlap in the measured DNA distribution between phases and due to inherent shortcomings in the mathematicals models, methods and techniques used.
It would, therefore, be desirable to have a relatively simple technique for completely resolving the DNA distributions in the three major phases of the cell cycle, G.sub.1, S and G.sub.2 M.